The following definitions are provided to facilitate an understanding of the present invention.
The term "target" or "target molecule" in a diagnostic sense, refers to a molecule of interest, i.e. the molecule whose presence one wishes to know. In a therapeutic sense, the term "target" or "target molecule" refers to a molecule associated with a disease.
The term "biological binding pair" as used in the present application refers to any pair of molecules which exhibit mutual affinity or binding capacity. A biological binding pair is capable of forming a complex under binding conditions. For the purposes of the present application, the term "ligand" will refer to one molecule of the biological binding pair, and the term "antiligand" or "receptor" will refer to the opposite molecule of the biological binding pair. For example, without limitation, embodiments of the present invention have application in nucleic acid hybridization assays where the biological binding pair includes two complementary nucleic acids. One of the nucleic acids is designated the ligand and the other nucleic acid is designated the antiligand or receptor. One of the nucleic acids may also be a target molecule. The designation of ligand or antiligand is a matter of arbitrary convenience. The biological binding pair may include antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates, to name a few.
The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand or receptor. As applied to nucleic acids, the term "probe" refers to nucleic acid having a base sequence complementary to a target nucleic acid. The probe and the target are capable of forming a probe target complex under binding conditions. The term "probe" will be used herein, in both a diagnostic sense, meaning capable of binding a molecule, the presence or absence of which one desires to know, and a therapeutic sense, capable of binding to a molecule associated with a disease.
The term "label" refers to a chemical moiety which is capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes. The term "agent" is used in a broad sense, including any chemical moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any chemical moiety which participates in reactions with the label.
The term "active" and "inactive" are used in a relative sense. The term "active" suggests normal or optimal chemical biological activity or reactiveness, and also encompasses such biological activity or reactiveness which, although less than normal or optimal, is greater than some other level of activity or reactiveness. The term "inactive" suggests exhibiting less biological activity or reactiveness than active.
The term "amplify" is used in the broad sense to mean creating an amplification product, which may include by way of example, additional target molecules, or target-like molecules, capable of functioning in a manner like the target molecule, or a molecule subject to detection steps in place of the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a polynucleotide, additional target, or target-like molecules, or molecules subject to detection can be made enzymatically with DNA or RNA polymerases.
The term "ribozyme" refers to an RNA structure of one or more RNAs having catalytic properties. Ribozymes generally exhibit endonuclease, ligase or polymerase activity.
The term "contiguous" means an adjacent area of a molecule. By way of example, in the case of biological binding pairs, where a first ligand binds to a receptor target molecule, the area surrounding and adjacent to the first ligand is open and capable of binding to a second ligand contiguous to the first. In the context of nucleic acid, where a first probe binds to an area of a larger nucleic acid target molecule, an adjacent mutually exclusive area along the length of the target molecule can bind to a second probe which will then be contiguous to the first. The target molecule acts as a template, directing the position of the first probe and the second probe. The term "substantially contiguous" is used in the functional sense to include spatial orientations which may not touch, may not abut, or may overlap, yet function to bring parts, areas, segments and the like into cooperating relationship.
The term "autocatalytically replicatable" refers to enzymatically catalyzed, self-directed replication of the type characterized by several RNAs and RNA enzymes. By way of example the enzyme, RNA-dependent RNA polymerase, of the bacteriophage Q-Beta (Q-Beta replicase), under reaction conditions, will act on a 221 nucleotide RNA template, known generally as midivariant-1 (MDV-1), and variations of MDV-1, including without limitation, minivariant RNA, microvariant RNA, nanovariant RNA, and modifications thereof to produce many copies of the RNA template. Other enzymes which participate in autocatalytic replication processes are, without limitation, SP replicase and MS2 replicase.
The term "capture ligand" means a ligand capable of specifically binding with a capture antiligand associated with a support.
The term "retrievable support" is used in a broad sense to describe an entity which can be substantially dispersed within a medium and removed or separated from the medium by immobilization, filtering, partitioning, or the like.
The term "support," when used alone, includes conventional supports such as filters and membranes as well as retrievable supports.
The term "reversible," in regard to the binding of ligands and antiligands, means capable of binding or releasing upon imposing changes which do not permanently alter the gross chemical nature of the ligand and antiligand. For example, without limitation, reversible binding would include such binding and release controlled by changes in pH, temperature, and ionic strength which do not destroy the ligand or antiligand.
Genetic information is stored in living cells in thread-like molecules of DNA. In vivo, the DNA molecule is a double helix of two complementary strands of DNA, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding and .pi.-stacking interactions. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand. The genetic code of a living organism is carried upon the DNA strand, in the sequence of base pairs.
Molecules of DNA consists of covalently linked chains of deoxyribonucleotides and molecules of RNA consists of covalently linked chains of ribonucleotides. Each nucleic acid is linked by a phosphodiester bridge between the 5'-hydroxyl group of the sugar of one nucleotide and the 3'-hydroxyl group of the sugar of an adjacent nucleotide. The terminal ends of nucleic acid are often referred to as being 5'-terminal or 3'-termini in reference to the terminal functional group. Complementary strands of DNA and RNA form antiparallel complexes in which the 3'-terminal end of one strand is oriented and bound to the 5'-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the tendency of two nucleic acid strands to pair at their complementary regions to form hybrids. The formation of such hybrids can be made to be highly specific by adjustment of the conditions (sometimes referred to as stringency) under which this hybridization takes place such that hybridization will not occur unless the sequences are precisely complementary. If total nucleic acid from the sample is immobilized on a solid support such as a nitrocellulose membrane, the presence of a specific "target" sequence in the sample can be determined by the binding of a complementary nucleic acid "probe" which bears a label. After removal of non-hybridized probe by washing the support, the amount of target is determined by the amount of detectable moiety present.
The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples, may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility, cancer and precancerous states, or bacterial or viral infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures may indicate the presence of antibiotic resistance, toxicants, viral- or plasmid-born conditions, or provide identification between types of bacteria. Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agriculture and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxicant-producing bacteria.
However, the sensitivity of such assays is limited by the number of labelled moieties which one may physically incorporate into the probe nucleic acid. In the case of radioactively-labelled probes, the practical limit of deletion is about 10.sup.4 target molecules. To achieve this sensitivity requires probes radioactive labels which have a very high energy and a very limited useful lifetime. The detection step, autoradiography, requires several days. Other labelling methods utilizing fluorescent, chemiluminescent, or enzymetic detection, although more rapid, usually do not exceed the sensitivity of radioactively-labelled probes. Since most organisms of clinical interest do not contain more than 50,000 copies of any nucleic acid suitable for use as a target, the utility of such methods is restricted to the detection of large numbers of organisms. The level of infectious agents in clinical specimens or foodstuffs, however, often does not exceed one to ten organisms.
One approach for the detection of low levels of DNA utilizes a DNA-dependent DNA polymerase to directly replicate the DNA target to increase its numbers to easily detectable levels. This approach is termed "polymerase chain reaction" (PCR). Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N., "Enzymatic Amplification of Beta-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia," Science 230: 1350-1354 (1985); Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A., "Primer-directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase," Science 239: 487-491 (1988); Erlich, H. A., Gelfand, D. H., and Saiki, R. K., "Specific DNA Amplification: Nature 331:461-(1988) and Mullis et al., European Patent application Nos. 200362 and 201184 (see also U.S. Pat. Nos. 4,683,195 and 4,683,202).
In practice, PCR is limited by the requirement that the target for amplification be DNA (as opposed to RNA), and by the occurrence of false positives generated by hybridization of probes to homologous sites in non-target DNA which fortuitously generate similar replication products. Moreover, although target DNA may be detected with very high sensitivity, the numbers of targets present in the sample is difficult to determine without adding significantly to the complexity of the assay. Since the number of infectious agents is often important in evaluating the treatment protocol for disease, this amplification approach is disadvantageously limited because it provides qualitative rather than quantitative results.
Another approach to improving the sensitivity of nucleic acid detection is to employ a nucleic acid probe associated with an autocatalytically replicatable RNA molecule. As used herein, the word "associated" means linked to or incorporated within. For example, a number of means to generate RNA probes by derivatizing MDV-1 RNA, a template for Q.beta. replicase, are suggested by Chu, B. C. F., Kramer, F. R., and Orgel, L. E., "Synthesis of an Amplifiable Reporter RNA for Bioassays", Nucl. Acids Res. 14: 5591-5603 (1986); Lizardi, P. M., Guerra, C. E., Lomeli, H., Tussie-Lune, I, and Kramer, F. R., "Exponential Amplification of Recombinant-RNA Hybridization Probes', Bio/Technology 6:1197-1203 (October, 1988); and European Patent Application 266,399 (EP Application No. 87903131,8).
An autocatalytically replicatable RNA-probe construct may be employed in a sandwich hybridization assay, such as that described by Ranki, et al., U.S. Pat. No. 4,563,419; Soderlund, G. B., U.S. Pat. No. 2,169,403; Stabinsky, U.S. Pat. No. 4,751,177; and Syvanenen, et al., Nucl. Acids Res. 14:5037-5048. In the event the target is present and probe has hybridized to target, the autocatalytically replicatable RNA associated with the probe is replicated to generate amounts of RNA which may be easily detected by a variety of means (for example, by fluorescence using a dye such as ethidium bromide or propidium iodide). Since the MDV-1 RNA template for Q.beta. replicase is doubled in number every 20 seconds in vitro, an exponential increase (estimated to be a billion-fold) in the number of RNA molecules occurs within a few minutes at a single temperature. The autocatalytic reaction proceeds at an exponential rate until the number of autocatalytically replicatable RNA molecules exceeds the number of active enzyme molecules in the reactions. After that point, the amount of autocatalytically replicatable RNA increases linearly with time. As a consequence, in reactions given a sufficient period of time to reach this linear phase (for example 15 minutes for 100 molecules), the amount of amplified product RNA will be directly related to the logarithm of the number of autocatalytically replicatable RNAs initially added (Lizardi et al., supra). Since the initial number of autocatalytically replicatable RNA probes is proportional to the amount of target, the amount of target present in the sample being examined may be quantitated over a very wide range.
Autocatalytic replicatable RNA probe constructs have been suggested in the art. In one approach, the probe is coupled to the RNA via a cystamine moiety containing a disulfide (--S--S--) linkage which can be cleaved prior to replication (Chu et al., supra). However, this method suffers from the need for several synthesis steps that increase the cost in labor of producing such probes. In addition, disulfide linkages are subject to premature cleavage by reducing agents (for example, glutathione) which occur naturally in many biological samples.
In another approach, the probe sequence may be incorporated within the sequence of the replicatable RNA (Lizardi et al., supra). However, the probe sequence is viewed as foreign by the enzyme and affects the ability of the RNA to be efficiently replicated, or is spontaneously deleted during replication. Deletion events affect the rate of replication and occur randomly with time. When deletion events occur, the level of the RNA products obtained in the linear phase of the amplification cannot be used to assess target level.
Linking the probe sequence to either the 3' or 5' termini of autocatalytically reproducible RNAs via the phosphodiester linkage normally found in RNAs, although simple to accomplish by a variety of means, has been reported to strongly inhibit replication. For example, ligation of a short oligoribonucleotide, A.sub.10, to the 5' nucleotide of MDV-1 RNA rendered the RNA unable to replicate exponentially (Miele, Ph.D. thesis, Columbia University, 1982). Attachment of additional nucleotides to the 3' terminus of other autocatalytically reproducible RNAs similarly inhibits their replication by Q.beta. replicase. For example, addition of between 10-20 cytidylate residues to the 3' terminus of Q.beta. phage RNA abolishes its template activity (Gillis, E., Devos, R. and Seurinck-Opsomer, C., Arch. Int. Physiol. Biochem. 84:392-393 (1976)); addition of a short oligoadenylate tract has a similar effect (Gilvarg, C., Jockusch, H. and Weissmann, C., Biochem. Biophys. Acta 414, 313-8 (1975); see also Devos, R., van-Emmelo, J., Seurinck-Opsomer, C., Gillis, E., and Fiers, 14., Biochim. Ciophys. Acta. 447:319-27 (1976)). Chu et al. in WO 87/06270 suggest, that attachment of an affinity molecule might be possible to autocatalytically reproducible RNA provided it is done through a purine linkage. The purine linkage would be subjected to an acid depurination cleavage procedure prior to replication. The clear implication of the Chu application, which is consistent with all the other teachings in the art, is that the autocatalytically reproducible RNA bearing terminally added sequences is inactive until cleaved.
The discussion thus far has focused on signal generation. Signal generation which is related to the presence of target is very desirable. Signal generation which is not related to target, referred to as background is undesirable. By way of example, a single autocatalytically replicatable RNA molecule in the presence of Q-Beta replicase and reaction conditions, will initiate the production of copies at an exponential rate. In the event such single autocatalytically replicatable RNA is associated with a probe, which probe is bound to target, the exponential replication is a true positive detection. In the event such single autocatalytically replicatable RNA is not associated with a probe, or if associated with a probe and such probe is not associated with target, the exponential replication is a false positive or constitutes background from which true signal must be differentiated. The presence of background limits the sensitivity of assays at low target concentrations. Target induced signal must be significantly greater than background in order for assays to be considered reliable.
One form of background, in affinity assays, occurs when the probe having a label associates with molecules other than target, and is carried through to detection. This type of background is often associated with non-specific binding of probe to supports.
One approach to reducing this non-specific binding background employs a method by which the target-probe complex is reversibly bound to the support ("reversible target capture"). After hybridization and immobilization, the complex is eluted from the support, which is then discarded with the non-specifically bound probe. The target-probe is then recaptured on fresh support. This process may be repeated several times to produce a significant reduction in the amount of non-hybridized probe (see Collins, European Patent Application No. 87309308.2).
A further type of background, common with autocatalytic replicatable amplification systems, is "unprimed" activity of the enzyme itself. Prior to the advent of purified Q-beta replicase, it was believed that the enzyme inherently had the capability to create MDV-1, without a template.
In a therapeutic sense, the ability to activate nucleic acid in a controlled manner is useful to control the expression of genes or to remove cells which harbor infection. By way of example, the control of viral genes with antisense molecules can prevent viruses from replication. In the alternative, cells which harbor viruses can be poisoned by autocatalytically replicating RNA to prevent viruses from infecting other cells.
The inability to control the amplification of autocatalytically replicatable molecules for diagnostic and therapeutic purposes has limited the application of such technology.